First published online 1 November 2006
doi: 10.1242/dev.02658
Development 133, 4737-4748 (2006)
Published by The Company of Biologists 2006
Grainyhead-related transcription factor is required for duct maturation in the salivary gland and the kidney of the mouse
Yoshifumi Yamaguchi1,2,*,
Shigenobu Yonemura3 and
Shinji Takada1,4,
1 Okazaki Institute for Integrative Biosciences, National Institutes of Natural
Sciences, Myodaiji, Okazaki, 444-8787, Japan.
2 Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto, 606-8502, Japan.
3 Laboratory for Cellular Morphogenesis, Center for Developmental Biology,
RIKEN, Kobe, 650-0047, Japan.
4 The Graduate University for Advanced Studies (SOKENDAI), Myodaiji, Okazaki,
444-8585, Japan.
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Fig. 1. CP2L1 expression in ducts of exocrine glands and kidney. (A,B,D-K)
LacZ expression from the Cp2l1 trap locus in the duct of
submandibular and sublingual glands (A), isolated SMG (B),
parotid and lachrymal glands (D) and nasal gland (E) at E16; in
the glandular ducts in olfactory epithelium (F) and ducts of the
mammary gland (G), male reproductive system (I), endolymphatic
sac (J) and lung (K; right, Cp2l1+/tra;
left, wild-type control) at birth; and in eccrine glands in the palm of the
adult (H; right, Cp2l1+/tra; left, wild-type
control). (C) Co-detection of endogenous CP2L1 protein (magenta) by an
antiserum to CP2L1 (As2375) and keratin 7 (green) in the duct of the SMG.
(L) Genotyping of Cp2l1 trap locus mice. The gene-trap vector
pLSAßgeo was inserted into the second intron of the Cp2l1 locus.
Amplification primers were designed as shown in the lower portion of L. This
set of primers could distinguish between the wild-type allele (+) and the
Cp2l1 trap allele (tra). (M) Immunohistochemical detection of
CP2L1 protein in a kidney section using As2375. Although this serum reacted
with unidentified proteins in the cytoplasm of glomerulus cells, CP2L1 protein
in the nucleus was not detected in Cp2l1tra/tra mice.
(N) Survival rate of Cp2l1+/tra (n=56) and
Cp2l1tra/tra mice (n=48). (O) Growth curve
of each genotype. P0-P21, postnatal day 0-21. Scale bar: 25 µm in C,M; 100
µm in J. LG, lachrymal glands; PG, parotid glands.
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Fig. 2. Morphological abnormality in the submandibular duct of
Cp2l1tra/tra mice. (A) Schematic representation
of the rodent SMG. (B) Morphological abnormalities observed in adult
male and female Cp2l1tra/tra mice. (C-E) In situ
hybridization analysis of the adult SMG. Nuclei were counterstained with
nuclear Fast Red (red). The GCT marker ß-NGF (C) and the basal
cell marker Sgn1 (D) were not detected in either male or female
Cp2l1tra/tra mice. Mist1 expression was normal in
the mutants (E). (F) Hematoxylin/eosin-stained sections of wild-type
(+/+) and Cp2l1tra/tra (tra/tra) fetuses at E18. Although
the numbers of cells surrounding the lumen of Cp2l1tra/tra
was almost equal to that of Cp2l1+/tra mice [mean cell
number ± standard deviation, 9.0±1.7 (n=25) versus
8.8±1.8 (n=20) in intralobular ducts and 10.8±1.7
(n=20) versus 10.2±1.8 (n=16) in the excretory
ducts], the lumen of Cp2l1tra/tra was significantly wider
in Cp2l1+/tra than in Cp2l1tra/tra
mice [mean diameter ± standard deviation, 2.69±0.83
(n=25) versus 3.68±1.16 µm (n=20) in the
intralobular ducts (P<0.005) and 5.34±2.77 (n=20)
and 7.2±2.0 µm (n=16) in the excretory ducts
(P<0.05)]. (G,H) Electron microscopic observations
of the duct. Apical structures of luminal cells (arrowheads in G and arrows in
H) did not develop sufficiently in Cp2l1tra/tra compared
with Cp2l1+/tra mice. (I) Immunohistological
staining of CP2L1 and keratins in the SMG at birth. Anti-CP2L1
immunoreactivity was found only in the duct cells (left panels). Detection of
cytokeratins (right panels) on the luminal surface in both the ducts (white
arrows) and acini (asterisks) in Cp2l1+/tra mice. In
Cp2l1tra/tra mice, cytokeratins were detected in acini but
not in the ducts. Scale bars: 100 µm in B-E; 10 µm in F; 7 µm in G; 1
µm in H; 50 µm in I. BC, basal cell; EXD, excretory duct; ID,
intercalated duct; STD, striated duct.
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Fig. 3. Abnormalities in gene expression in the SMG of
Cp2l1tra/tra mice. (A-D,G) In situ
hybridization analysis of the SMG at birth. Expression of the duct-specific
genes keratin 7 (A), Gk-6 (B), Fxyd2b (C), Scnn1b
(D) and keratin 19 (G) was abnormal in Cp2l1tra/tra mice
compared with Cp2l1+/tra mice. (E) Expression of
CP2L1 and keratin 7 examined in adjacent sections of the nasal gland. In the
duct of the nasal gland, Cp2l1 (lacZ expression; left) was
coexpressed with keratin 7 (right) in control Cp2l1+/tra
mice (upper panels). In Cp2l1tra/tra mice (lower panels),
based on the morphology and the expression of Cp2l1 (left), the duct
was formed, although keratin 7 (right) was not expressed. (F)
Comparison of mRNA expression levels in the newborn SMG of
Cp2l1+/tra and Cp2l1tra/tra mice by
quantitative RT-PCR. *P<0.005. Scale bars: 100
µm.
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Fig. 4. CP2L1 expression during kidney development. (A) Detection of
CP2L1 protein (green) in the ureteric epithelium (CD; arrowhead), identified
by expression of cytokeratin (red), and at the distal end of the S-shaped body
(arrow) at birth. DNA was stained with TOPRO3 (blue). (B) CP2L1
(lacZ) was detected strongly in the CNT, DCT and TAL and weakly in
the DL and PCT at the primitive loop nephron at E16. (C-F)
Identification of CP2L1 expression domains at birth by co-labeling by
LacZ staining (blue) and in situ hybridization (orange). Insets:
higher magnification of boxed regions. CP2L1 (LacZ) was strongly
coexpressed with Ncx1 (C) and modestly or weakly with Ncc
(D) at the immature loop nephron in deeper regions of the cortex. Few CP2L1
were detected with Nkcc2 (E) (arrowheads) and Slc34a1 (F).
Note that RNA localization of several genes, including Slc34a1, Ncx1
and Ncc (C,D,F), was detected only in the nucleus by in situ
hybridization used in this analysis. (G) Double-labeling of CP2L1
(lacZ) and Umod (orange) in the medulla. The expression of
CP2L1 was weakly detected in Umod-positive TAL at P0 (blue dots
indicated by black arrowheads in the upper inset) but disappeared in
adulthood. (H) Expression of CP2L1 (lacZ) at P0 and P10. The
expression was lost at P10 in the papillary region (white arrows). (I)
Summary of CP2L1 expression. The expression shifts during the course of
nephron maturation. Scale bar: 20 µm in A; 50 µm in C-G; 200 µm in
H.
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Fig. 5. Kidney formation in Cp2l1tra/tra mice.
(A,B) PAS staining of a kidney section at postnatal day 2.
Higher magnification figures of A are shown in B. (C) Expression of
AQP2 protein in the medulla was normal in both newborn
Cp2l1+/tra and Cp2l1tra/tra mice.
(D) Expression of Pax2 in CD (arrowheads) was normal even in
Cp2l1tra/tra mice at birth. (E) Quantification of
mRNA expression levels of nephron segmental marker genes at birth by
quantitative RT-PCR. Values represent mean expression levels ± standard
deviation (n=4-9). The experiments were repeated at least twice, and
there was no significant difference between Cp2l1+/tra and
Cp2l1tra/tra mice. Scale bars: 200 µm.
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Fig. 6. Differentiation defects in the kidney of
Cp2l1tra/tra mice. (A-K) In situ hybridization
analysis of the kidney at birth. Expression of IC markers Atp6b1 (A)
and Slc4a1 (B) was lost in Cp2l1tra/tra mice.
Expression of keratin 7 was lower in the whole CD of
Cp2l1tra/tra mice at E18 (C,E) and in the cortical CD
(D,F; arrowheads) of newborn Cp2l1tra/tra mice. By
contrast, keratin 19 was more strongly expressed in the inner medullary CD of
Cp2l1tra/tra mice than Cp2l1+/tra mice
at birth (G). Expression of Gk-6 and Fxyd2c (orange) in DCT
and CNT overlapped with that of Cp2l1 (lacZ staining; blue)
(H). Expression of Gk-6 and Fxyd2c was not detected in
Cp2l1tra/tra mice at E18 (I,J). Expression of
Clcnkb was also absent in the distal tubules of
Cp2l1tra/tra mice (K). (L,M) Quantification
of mRNA expression by quantitative RT-PCR in isolated E14 kidney organ
cultures (L) or in dissected kidneys at different developmental stages (M).
The experiments were repeated at least twice. *P<0.005,
P<0.05. (N) Comparison of gene
expression between the kidney and the salivary glands. Analogous changes in
gene expression were observed in these organs in
Cp2l1tra/tra mice. Changes are indicated with arrows, and
a battery of genes commonly expressed in the developmental programs of these
organs is shown in color. Scale bars: 200 µm in A,C,D,G,I,K; 100 µm in
B,E,F,H,J.
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